With the increasing interest in environmental, clinical and other biological problems, there is a growing need for trace analytical methods that are suitable for complex organic mixtures. While gas chromatography, particularly in combination with mass spectrometers, has been successfully applied to the volatile species, high-performance liquid chromatography (HPLC) is many times used for the non-volatile components. Although HPLC technology has made big gains recently, the overall separatory power is still not competitive with gas chromatography. Therefore, there is a real need for improved HPLC detectors, since small sample sizes are required for the highly efficient HPLC separations. Thus, the detectors could be improved with respect to their detectabilities.
Of the three most commonly used HPLC detectors, the fluorometric detectors has been developed sufficiently to be suitable for most situations. For non-fluorescing samples, the absorption detector must be used, but the detection of small differences in two large signals limits conventional detectors to the 10.sup.-3 to 10.sup.-4 range in absorbence. When the species of concern does not show convenient absorption bands, e.g., saturated organic compounds, the refractive index detector is commonly used, despite its poor sensitivity. Since the scope of application of HPLC is virtually related to the detectability of the detectors, there is a real need to improve the refractive index and absorption detectors in sensitivity and detectability.
Absorption detectors can be improved by monitoring an associated effect other than the decrease in light intensity. The most convenient associated effect is the generation of heat through relaxation of the excited molecules of the specimen. The non-uniform heating resulting from absorption of a laser beam gives rise to thermal lens calorimetry. If instead, the temperature gradient, and thus a refractive index (RI) gradient, that is developed is used to deflect a probe laser beam, the technique of photothermal deflection is created. One can also use the heat waves that are generated by a pulsed or chopped excitation source as the basis for photoacoustic detection. These concepts have already been demonstrated as a detection scheme for HPLC. Since the magnitudes of all of these associated effects increase with the power of the excitation (absorbed) light source, one can achieve lower detectabilities by using these associated effects as compared to conventional measurements.
Perhaps the most sensitive way to monitor small changes in the refractive index is interferometry. The same technology that allows one to achieve high frequency stability in lasers and to measure these frequencies to great precision and accuracy, can be applied to the detection of refractive index changes.
One method of doing so would be to use a Mach-Zender interferometer and a single frequency laser to monitor the phase delays in a sample due to absorption and the subsequent heating to detect trace gases. This type of phase-fluctuation heterodyne spectroscopy has been shown to be a fairly good gas chromatography detector. However, detectability depends upon the quality of the interference that can be achieved. The Mach-Zender interferometer has relatively low finesse because a low reflectability mirror (50%) is used for splitting the beam into two paths. It also suffers from having an "idle" arm that can be perturbed by acoustic waves unless the system is evacuated.
Further, arrangement of the optical components is such that system rigidity is difficult to maintain. The Fabry-Perot interferometer, on the other hand, typically has very high finesse, has no "idle" optical paths, and is commercially available with excellent rigidity using materials with low co-efficients of thermal expansion such as Super-Invar, a special composition made principally of nickel and iron and available from such companies as Guterl of Lockport, N.Y.
Additionally, detectability of traditional absorption detectors in HPLC needs improvement. Traditional absorption measurements gain linearly with increasing interaction length. In interferometry, increased length increases the absorbed amount, but at the same time, more volume must be heated up. Therefore, there is no net gain in the accuracy of the change in refractive index. However, the resolution of the interferometer generally increases with the distance between its mirrors, so that longer light paths are still desirable.
Therefore, it is an object of this invention to provide a refractive index and absorption detector which successfully adapts Fabry-Perot interferometry to high performance liquid chromatography detection in a system that allows the improved detectabilities of both the refractive index and absorption.
A further object of this invention is to provide a refractive index and absorption detector which provides superior measurements to traditional HPLC detectors.
A further object of this invention is to provide a refractive index and absorption detector which can provide orders-of-magnitude improvement in detectability over commercial HPLC refractive index detectors.
A yet further object of this invention is to provide a refractive index and absorption detector which achieves a detectability orders-of-magnitude better than standard absorption detectors in HPLC.
A further object of this invention is to provide a refractive index and absorption detector which has a very high finesse, has no "idle" optical paths, and is commercially available with excellent rigidity using materials with low co-efficients of expansion.
Another object of this invention is to provide a refractive index and absorption detector which improves the accuracy of both in the same instrument.
A further object of this invention is to provide a refractive index and absorption detector which is economical, durable, accurate and easy to use.
Additional objects, features and advantages of the invention will become apparent with reference to the accompanying specification and drawings.